Our long-term goal is to understand the mechanism of protein sorting and recycling from vesicular tubular clusters (VTCs). VTCs function as transport complexes delivering cargo from the ER to the Golgi complex. Moreover, these structures are the first site of segregation of the anterograde and retrograde pathways and thereby perform a critical function. The small GTPase Rab2 plays an essential role in this trafficking step by promoting formation of retrograde-directed vesicles that carry recycling cargo back to the ER. Rab2- mediated vesicle formation requires atypical PKCi/X and GAPDH. In this application we propose to characterize Rab2 mechanistically and to establish the role of Rab2 in events associated with VTCs. The Specific Aims are: 1) to identify the protein components of the VTC subcompartment that binds Rab2, 2) to characterize the interaction between components of the Rab2 complex by mapping their reciprocal binding domains, and 3) elucidate the molecular mechanism of Rab2-dependent-retrograde vesicle budding from VTCs. The importance of investigating Rab2 function in secretion is underscored by the observation that elevated Rab2, PKC, and GAPDH expression correlates with high metastatic potential in various cancers. Since metastasis is a reflection of cytoskeletal reorganization and increased cell motility, it is noteworthy that Rab2 and its effectors influence microtubule dynamics. However, the precise function of Rab2, aPKCi/A, and GAPDH in the early secretory pathway and how these proteins contribute to the disease are unknown. The studies proposed in this application will develop a new and novel paradigm that couples Rab2-Apkc lA-GAPDH dysfunction with pathological consequences.